Domains required for the interaction of the central negative element FRQ with its transcriptional activator WCC within the core circadian clock of Neurospora

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ–FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC–WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC–FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC–WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC–WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length.

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length.
Circadian rhythms control a wide range of cellular and behavioral processes in most eukaryotes and certain prokaryotes (1,2), facilitating adaptation of life to constant environmental changes; disruption of circadian rhythms has been implicated in various diseases in humans (3). At the molecular level, circadian clocks rely mainly on interlocked positive and negative arms, and in circadian cycles, the latter gradually repress their own expression through inactivating the former on a timescale of hours. In the clock of Neurospora crassa, a circadian model organism used for decades, the White Collar complex (WCC) derived from all cellular White Collar-1 (WC-1) and a fraction of WC-2 drives transcription of the central pacemaker gene, frequency (frq), through binding to either of two DNA elements in the frq promoter under contrasting illumination conditions: either the Clock box (C-box) in the dark or the proximal light-response element upon light exposure or in constant light (4)(5)(6). FREQUENCY (FRQ), encoded by the frq gene, associates with FRH (FRQ-interacting RNA helicase) (7-9) and CK1 (casein kinase 1) (10) to create the FFC (FRQ-FRH complex), repressing WCC transcriptional activity by promoting its phosphorylation at a group of residues: S971, S988, S990, S992, S994, and S995 of WC-1 as well as S331, T339, S341, S433, and T435 of WC-2 (10-12) and thereby terminating its own expression to conclude a circadian cycle.
An orthologous feedback loop is found in mammalian cells, with CLOCK and BMAL1 forming a heterodimer that plays the role of WCC, driving expression of PERIODs (PERs) and CRYPTOCHROMEs (CRYs) which form a negative element complex with CK1, analogous to the FFC, that inactivates CLOCK/BMAL1 by phosphorylation at key residues (13,14). As is the case in Neurospora, while much is becoming known about the means through which repression is achieved (14)(15)(16)(17)(18), relatively little is known about the structural determinants on PERs/CRYs or CLOCK/BMAL1 that determine their interactions.
In Neurospora, WCC acts as a responsive hub coordinating the circadian clock that is obligate in constant darkness and light signaling (19). In consonance with its dual role as a light sensor and a transcription factor for frq in the clock, WC-1 bears a light-, oxygen-, and voltage-sensing domain, a transactivation domain, two motifs required for DNA binding-the zinc finger (ZnF) and its nearby DBD (defective in DNA binding) motif, and two Per-Arnt-Sim (PAS) domains for WC-2 interaction (20,21); WC-2, an accessory of WC-1, contains a PAS and a ZnF DNAbinding domain (20). The DBD motif (KKKRKRRK) on WC-1 has been discovered to be indispensable for WCC to bind the C box and to drive frq transcription in the dark as well as for interacting with FRQ/FRH (21).
FRQ functions mainly as an organizing platform for recruiting and scaffolding the circadian negative elements FRH and CK1 in forming the FRQ-FRH-CK1 complex which controls the pace of the clock (7,22). Self-interaction among FRQ molecules occurs via their coiled-coil domains and disruption of the coiled-coil results in arrhythmicity (23). The nuclear localization signal of FRQ is necessary for its circadian function in WCC repression (24). FRQ interacts with CK1 through two domains: FRQ-CK-1a interaction domain 1 (25) and FRQ-CK-1a interaction domain 2 (10) and with FRH via the FRQ-FRH interaction domain (7,26). FRQ also contains two PEST-like elements: PEST-1 and PEST-2, both of which undergo CK-1a-and CK-1b-mediated progressive phosphorylations, and elimination of PEST-1 results in arrhythmic conidiation, reflecting an impaired clock (27). Intramolecular interplay between the N and C termini of FRQ has been shown to be key for the clock operation (25).
Multisite phosphorylation has been investigated extensively as a major mechanism for fine-tuning circadian activities of both FRQ (10,(28)(29)(30) and its transcriptional factor WCC (10)(11)(12)31). Many phosphorylation isoforms of FRQ have been reproducibly seen in Western blotting from cultures grown in the light (32). As the initial step in feedback-loop execution, negative elements need to interact physically with the positive factors in order to promote posttranslational modifications to the latter and thereby bring about timely but gradual repression. While prior work on the Neurospora clock has focused on phosphorylation and transcription-centered mechanisms controlling the clock, it remains largely unknown how molecular contacts between the positive and negative element complexes are accomplished, though this step is plainly required for subsequent negative feedback inhibition. We previously uncovered a motif on WC-1 composed of eight consecutive highly basic residues required for interaction with FRQ (21), raising the corresponding question of which regions and residues of FRQ are reciprocally involved in the establishment of FFC-WCC. To tackle this puzzle, here we have characterized key regions of FRQ essential for the WCC-FFC interaction and also the operation of the core circadian oscillator. As biochemical data hinted that FFC and WCC may contact via electrostatic charges, we focused on negatively charged residues falling in the important regions of FRQ that determine WCC interaction. To this end, mutagenetic analyses revealed three clusters of FRQ residues that contribute to WCC association. Interestingly, however, the core clock still runs normally in certain frq mutants with an extremely low level of FFC-WCC, revealing that while interaction between the positive and negative elements is required for the circadian feedback mechanism, it is not a reliable measurement for period length.

Results
A frq deletion series identifies regions required for the Neurospora clock Three distinct regions on FRQ have been implicated in WCC interaction: amino acids 107 to 310, 435 to 558, and 631 to 905 (26). The loss of FRQ-WCC interaction in frq mutants missing any of these elements (26) might be attributed to the removal of the WCC-interacting domain(s) or pleiotropic effects caused by polypeptide shortening. To address this, we engineered a set of frq mutants each missing 50 amino acids situated in these regions. In these mutants, frq transcription as reported by a C-box-driven luciferase gene whose product was measured in real-time (Fig. 1). frq Δ564-603 shows a wildtype (WT) period length; frq Δ691-732 displays a shortened period of 15.5 h; frq Δ107-148 , frq Δ435-481 , frq Δ604-646 , frq Δ874-884 , and frq Δ857-905 have prolonged rhythms to variable extents. The core clock became totally arrhythmic in frq Δ149-193 , frq Δ194-199 , frq Δ200-249 , frq Δ250-310 , frq Δ482-510, frq Δ511-558 , frq Δ647-690 , frq Δ733-774 , frq Δ775-800 , and frq Δ801-856 . The data support the idea that multiple regions of FRQ take part in the rhythmicity control and period determination, as has been documented by a large body of literature.

FRQ-WCC integrity is affected in most arrhythmic strains
To examine the FFC-WCC formation that functions as the initial step in the repression elicited by FFC on WCC, FRQ (tagged with V5H6) in the frq mutants whose circadian phenotypes were analyzed by the luciferase assay in Figure 1 was pulled down with V5 resin, and FRQ, FRH, WC-1, and WC-2 were blotted with protein-specific antibodies (Fig. 2). The diffuse band of FRQ derived from multisite phosphorylations has been observed characteristically (32) from all the frq mutants that were grown in the light and tested (Fig. 2). When FRQ was enriched by immunoprecipitation (IP) in frq Δ149-193 , frq Δ194-199 , frq Δ200-249 , frq Δ482-510, frq Δ511-558 , frq Δ647-690 , frq Δ733-774 , frq Δ775-800 , frq Δ801-856 , and frq Δ857-905 , WC-1 and WC-2 became undetectable relative to the WT, and all except frq Δ857-905 showed an arrhythmic clock and enhanced C-boxdriven reporter activity (Figs. 1 and 2). FRQ-WCC is almost completely lost in frq Δ857-905 , but the mutant strain still showed a rhythm with prolonged period length (Figs. 1 and 2), suggesting that a stable FFC-WCC supercomplex is not required for the execution of the circadian feedback loop. frq Δ107-148 , frq Δ564-603 , frq Δ604-646 , and frq Δ691-732 displayed weak FFC and WCC interaction but sustained weak rhythmicity (Figs. 1 and 2). This result confirms that multiple domains on FRQ contribute cooperatively to contact with WCC. FRH dissociates from FRQ in frq Δ733-774 , frq Δ775-800 , and frq Δ801-856 (Fig. 2), which may lead to the loss of WCC in the complex as noted previously for frq mutants frq 6B2 and frq 6B5 (26). This also reinforces that FRQ needs to form a complex with FRH in order to interact with WCC (7,8,26), presumably through constructing a proper quaternary structure of FFC. The clock in frq Δ733-774 , frq Δ775-800 , and frq Δ801-856 does not oscillate ( Fig. 1), consistent with the finding that the absence of FRH in the FFC abrogates feedback loop closure (7,8). frq Δ250-310 , frq Δ435-481 , and frq Δ874-884 have normal FFC-WCC interaction (Fig. 2). Interestingly, although the interaction of FFC and WCC in frq Δ250-310 seems unaffected, or even slightly stronger relative to WT (upper left in Fig. 2), the mutant is arrhythmic (Fig. 1), which might be due to an indirect impact of the deletion on the nearby FRQ-CK1 interacting domain (FCD-1) that has been shown to be essential for the clock (25). Collectively, the data here further characterize FRQ's regions involved in WCC interaction but suggest that the binding strength of FFC-WCC is not indicative of the period length.

FFC-WCC interaction can be disrupted by high salt
A basic stretch (KKKRKRRK) near the ZnF DNA-binding domain of WC-1 was previously identified as required for the WCC-FFC organization (21), which suggests that ionic charges may drive the complex formation. To confirm this hypothesis, the NaCl concentration in the standard proteinlysis buffer was raised from 137 mM that is used commonly in the biochemical analysis of FFC and WCC to 500 and 1000 mM, either WC-1 or FRQ was immunoprecipitated, and the four clock components followed subsequently by Western blotting. Following WC-1 IP, the enrichment of WC-2 was barely affected by high salts in the buffer, but FRQ and FRH were undetected when 500 mM or 1000 mM sodium chloride was used (Fig. 3A). Similarly, in a separate experiment with epitope-tagged FRQ instead, FRQ V5H6 as well as bound FRH were immunoprecipitated readily with V5 antibody regardless of salt titers, whereas WC-1 and WC-2 disappeared from the FFC when salt dosages rose (Fig. 3B). The data support that FFC and WCC associate with each other by virtue of electrostatic forces, which are subject to high salt disruptions in vitro, while WC-1 and WC-2 bind together presumably by hydrophobic effects via their PAS domains (26); likewise, both FRQ and FRH possess specified domains buttressing their interaction (26,33). Accordingly, interactions between FRQ and FRH as well as WC-1 and WC-2 are less prone to high ionic strength disruptions.

Luciferase analysis of frq D/E-to-A mutants
Based on the observations that the organization of WCC-FFC is susceptible to the ambient ionic strength (Fig. 3) and also that the basic motif of WC-1 is needed for the WCC-FFC connection (21), negatively charged residues, Asp (D)/Glu (E), in the key regions of FRQ (Figs. 1 and 2) were mutated individually or in clusters to alanines for the purpose of pinning . Luciferase assays of frq partial-deletion mutants grown at 25 C in the dark. Strains were synchronized at 25 C in the light overnight, and bioluminescence signals were tracked every hour by a CCD (charge-coupled device) camera after moving the strains to the dark at the same temperature. Three replicates (lines in different colors) were plotted for each mutant with the x-axis and y-axis representing time (in hours) and arbitrary units of the signal intensity, respectively. In this and subsequent figures, period length was calculated from three or more replicates and reported as the average ± the standard error of the mean (SEM). All frq mutants throughout this study were targeted to the native locus with a tandem V5 and 6× histidine (V5H6) tag at their C termini. FRQ, FREQUENCY.
down (tagged) FRQ's residues involved in WCC binding. In subsequent luciferase reporter analyses, four classes of rhythm alterations were noted in these frq D/E-to-A mutants (Fig. 4 , and frq E511A, D521A , respectively. The data of period changes here agree fairly well with the periodalternating pattern of frq mutants from a recent publication demonstrating that mutating phosphorylation sites in the N terminal and middle regions of FRQ results typically in prolonged period lengths, while elimination of C-terminal phosphorylation events always lessens the period length (28). In frq E801A, D802A, E805A, E809A , the level of FFC-WCC interaction is comparable to that in WT (Fig. 5), but the mutant strain was still arrhythmic in the luciferase assay (Fig. 4), suggesting that stable interaction between FFC and WCC is not sufficient for sustaining rhythmicity but instead transient but dynamic in vivo contacts between the two complexes may be required for the efficient and persistent inhibition of WCC, especially to the fraction of WCC that associates with the C box of the frq promoter.
The FFC-WCC establishment is impaired in certain frq D/E-to-A mutants To determine whether the FFC-WCC in the D/E-to-A mutants from Figure 4  FRQ's D/E residues essential for WCC interaction examined expression and interaction of the four core-clock components, FRQ, FRH, WC-1, and WC-2 by IP and Western blotting. FRQ was not detected in frq D149A, D150A, D156A, D157A and frq E835A and was only very weakly detected in frq E760A, E763A, D773A, D774A after enrichment (Fig. 5), suggesting that mutations of these four residues may significantly impact FRQ stability; this explains why they displayed arrhythmicity but high signal intensity in the luciferase analysis ( Fig. 4), basically mirroring the behavior of Δfrq or frh mutants (7,8,34). FRQ abundance in frq E200A, E202A, D207A drops drastically, which might lead to a reduction of FRH in the complex. The observations of little to no expression of FRQ along with diminished abundance of WCC in frq D149A, D150A, D156A, D157A , frq E200A, E202A, D207A , frq E760A, E763A, D773A, D774A , and frq E835A are nicely compatible with the "black widow model" proposing a negative correlation between the activity of transcription factors in transcription with their cellular abundance (8,11,35). Interestingly, WC-1 and WC-2 levels in the FFC-WCC in frq D664A, D667A , frq D862A, D866A, D867A, D869A, D870A , and frq D874A, D875A, E876A, E877A, E879A, E880A, E882A, E883A, D884A become dramatically reduced (can only be visualized with a long exposure in Western blotting) relative to WT, though FRQ and FRH in these strains interact normally with each other (Fig. 5). This result appears to be astonishing as the three frq mutants demonstrate an approximate WT period (Fig. 4), but it evidently suggests that the stability of the interaction between the positive and negative element complexes does not predict the period length and also suggests that FRQ-promoted phosphorylation of WCC can persist efficiently even with less WCC bound in the complex.
The third cluster of D/E on FRQ crucial for the WCC-FFC formation FRQ was completely undetectable in frq D149A, D150A, D156A, D157A , frq E760A, E763A, D773A, D774A , and frq E835A (Fig. 5). To probe the role of these D/E clusters, we made frq mutants bearing fewer mutations at these sites, or an E835D substitution, and monitored C-box activity and WCC-FFC establishment. FRQ's E183 and E187 were also included in the mutagenesis because they are located nearby D149, D150, D156, and D157. The clock in frq D149A, D150A , frq E760A, E763A , and frq E835D oscillates robustly with an approximate WT period, while frq D156A, D157A , frq E183A, E187A , and frq D773A, D774A completely lost rhythmicity (frq E183A, E187A only shows one peak) (Fig. 6A). FRQ expression in frq D773A, D774A is extraordinarily low (a faint band after IP) (Fig. 6B), explaining the arrhythmicity in the luciferase assay (Fig. 6A). WC-1 and WC-2 levels in FFC-WCC became greatly diminished in frq D149A, D150A , once again showing that the WCC-FFC abundance in the cell does not faithfully reflect the period length (Fig. 5). The loss of FFC-WCC in frq E183A, E187A and frq D773A, D774A (Fig. 6B) is consistent with abolished rhythmicity (Fig. 6A). These data reinforce the necessity of incorporating FFC into WCC for eliciting phosphorylation-induced repression. It is noteworthy that unlike frq E835A in which FRQ protein  accumulation was abolished (Fig. 5), frq E835D bears a vigorously oscillating clock as well as normal FRQ abundance and unchanged interaction of FRQ with WCC and FRH in comparison with WT (Fig. 6B), suggesting that the negative charge of E835 is the main contributor in promoting FRQ accumulation.

Expression and clock-components interaction in frq truncations
Certain residues on FRQ, including D773, D774, and E835, contribute significantly to the accumulation of the full-length protein (Figs. 5 and 6). We wondered whether this observation also applies to truncated variants of FRQ and whether retaining certain clusters of important D/E suffices for WCC recruitment. Hence, we made two sets of frq mutants each bearing abridged versions of FRQ, and these truncated FRQ segments together cover all the residues of the full-length FRQ twice: frq 1-558 and frq 559-989 , frq 1-310 , frq 311-628 , and frq 629-989 (Fig. 7A). As expected, frq 1-310 , frq 311-628 , and frq 629-989 totally lost rhythmicity in the luciferase assay (Fig. 7A). The FRQ level in frq 1-310 fell below our detection limit in Western blotting even after an enrichment by IP, while in other mutants, it appears to be comparable to that in WT (Fig. 7B). Although FRH complexed with FRQ as strongly in frq 559-989 and frq 629-989 as in WT, WC-1 and WC-2 in all of these mutants was undetectable above the background in Western blotting (Fig. 7B), indicating that preservation of individual D/E clusters is not sufficient for FFC-WCC formation.

Glu (E) substitution of FRQ's D660 phenocopies frq D660A
FRQ's residue D660 was found to be essential for FFC to recruit WCC and thereby for the negative feedback loop (36). To test whether retaining the negative charge of D660  . Luciferase assays of frq D/E-to-A mutants at 25 C in the dark. Synchronization of strains was done in the light at 25 C overnight, and light signals were tracked every hour by a CCD camera following the transfer of the strains to the dark at 25 C. Lines in different colors represent three replicates with the x-axis and y-axis displaying time (in hours) and the signal intensity (arbitrary units), respectively. Period length was determined from three replicates and shown as the average ± the SEM. All frq mutants bear a V5H6 tag at their C termini, targeting at the frq native locus. CCD, charge-coupled device; FRQ, FREQUENCY.
preserves the role of FRQ in the clock, we generated frq D660E and assayed it for C-box activity by luciferase analysis and WCC interaction by IP. To our surprise, frq D660E behaved as arrhythmically as frq D660A (Fig. 8A) and failed to rescue the loss of the FFC-WCC in frq D660A (Fig. 1). The data reveal that the negative charge of D660 is not the sole factor in determining the FFC-WCC formation and circadian rhythmicity, an observation that contrasts sharply with what we have obtained from frq E835A and frq E835D (Figs. 5 and 6), demonstrating the importance of the side-chain charge of E835 in promoting FRQ accumulation. The intramolecular interaction between the N and C termini of FRQ has been noticed (25). E835 may be involved in the binding of the C-terminal tail of FRQ with its N terminus.

Discussion
In Neurospora, the frequency gene encodes the central scaffolding protein FRQ that organizes the multicomponent negative arm of the core clock, and the cellular timing information has been encoded sophisticatedly and situated precisely in its expression and probably more importantly in its chemical modifications. Unfortunately, the complete FRQ structure, like that of its mammalian counterparts the PERs, remains unresolved due mainly to its low abundance in the cell and intrinsically disordered nature. This leaves many questions concerning the core-clock operation unsettled. For instance, how do the negative arm complexes FFC and PERs/CRYs accurately bridge kinases to progressively inhibit WCC or CLOCK/BMAL1 via phosphorylation in the repressive phase of the clock and how is it that FFC only binds to a small percentage of WCC but is able to proficiently repress all the WCC molecules in the cell. In this study, we attempted to probe these queries by searching for important regions and more specifically key residues of FRQ engaging in WCC contacts. Segment-deletion analysis identified regions on FRQ required for the core oscillator as well as WCC recruitment (Figs. 1 and 2). In agreement with prior literature, more than one region on FRQ contributes to WCC interaction (Fig. 2). Biochemical analysis further revealed that the FFC-WCC complex is vulnerable to elevated salt; the DBD on WC-1 was identified as a key for FFC interaction. Altogether, these leads guided us to dissect negatively charged residues falling in the regions of FRQ that were found to be important for WCC interaction (Fig. 1). In addition, we found residues vital for FRQ accumulation or stability as well as the ones significantly impacting period length when mutated (Fig. 4). IP assays confirmed that three clusters of negatively charged amino acids, D149/D150/D156/D157/E183/E187, D664/D667, and D/E from aa 862-D884 (Fig. 8B)  association, but surprisingly, frq D664A, D667A , frq D862A, D866A, D867A, D869A, D870A , and frq D874A, D875A, E876A, E877A, E879A, E880A, E882A, E883A, D884A displayed vigorous circadian rhythms with a period length almost identical to WT. These data indicate that the stability of the interaction between the positive and negative elements does not control the pace of the oscillator. It remains elusive whether the FRQ's D/E residues discovered in this study are located on the interface of the FRQ structure and contact the WCC directly or whether they contribute to the maintenance of FRQ's tertiary structures that are needed in WCC recognition. Recently, the binding strength of FRQ-CK1 has been demonstrated to be an indicator of the period length at normal temperatures as well as in a physiological temperature range (37,38). The correlation of WCC-FRQ abundance with period length is vague since frq mutants with a constant WCC-FRQ level showed varying period lengths (37). Most WCC in the cell resides in the nucleus, while the majority of FRQ is located in the cytoplasm (12,21,24,31). These observations underpin the fact that even in the WT strain, only a small portion of the FFC pool encounters and then complexes with WCC (12). The FFC-WCC supercomplex (in terms of size) (12) may assemble in a dynamic manner as evidenced by the quantitative proteomic data showing that WCC interacts preferentially with hypophosphorylated FRQ, whereas FRH associates constantly with FRQ independent of the latter's phosphorylation status. Unexpectedly, we here saw that the period length in certain frq mutants was almost unaffected even with the severely impaired WCC-FFC establishment based on co-IP. Based upon prior evidence, this may be interpreted in several ways. First of all, there may be only a limited fraction of the WCC pool actively participating in frq transcription, and correspondingly timely expressed FRQ only needs to repress this small amount of DNA-bound WCC. Thus, the core oscillator could operate in a normal way even in mutants composed of markedly less FRQ and WCC (similar to the scenario in WT with most FFC and WCC not participating in the core oscillator). This has been suggested by experimental data showing that downregulation of WCC does not greatly perturb circadian rhythmicity beyond a slight 2 h period lengthening (35). Second, the dynamic organization of WCC-FFC may ensure that the clock can be sustained with less WCC and FFC involved. Previous findings suggested that both WCC and FRQ rapidly translocate between the nucleus and cytoplasm (39,40). Finally, the course of FFC-mediated WCC repression via phosphorylation lasts more than roughly half a day (41,42). Therefore, FFC in certain mutants may be still fully capable of bringing about sufficient inactivation to WCC over a long while, even though the inactivation efficiency may be not as high as that in WT. These possibilities need not be mutually   Figure 6. FRQ residues D149, D150, D156, D157, E183, and E187 are needed for FFC to bind WCC. A, luciferase assays of indicated frq mutants at 25 C in the dark. Synchronization for the clock was carried out at 25 C plus light overnight, cultures were transferred to the dark, and then bioluminescence signals from the dark-grown strains (at 25 C) were tracked every hour. Three replicates are represented by differently colored lines with time (in hours) as the x-axis and the signal intensity (arbitrary units) as the y-axis. Period length was measured from three replicates and displayed as the average ± the SEM. All frq alleles were appended with a V5H6 tag at their C termini at the frq locus. B, interaction of clock components FRQ, FRH, WC-1, and WC-2 in the stated frq mutants. V5H6-tagged FRQ was immunoprecipitated with V5 resin from strains cultured in the light at 25 C for 24 h, and WB was carried out with indicated antibodies against FRQ, FRH, WC-1, or WC-2 (see Experimental procedures for details). The red box denotes remarkably decreased or undetected WCC from the FRQ pull-down (by V5 exclusive, including that FFC may only need to repress DNAbound WCC, and this process may proceed in a slow manner in vivo. Most likely, transient interaction between the two complexes may be sufficient for their recognitions and also for FFC-mediated repression of WCC via phosphorylation. Of course, the transient interaction might be affected in biochemical analyses, likely even from the point of the breaking up of a cell. FRQ phosphorylation has been extensively explored for decades as the primary regulatory mechanism governing the Neurospora clock (10,(28)(29)(30)(43)(44)(45)(46)(47)(48)(49)(50). Newly synthesized hypophosphorylated FRQ possesses higher affinity for WCC than the massively phosphorylated isoforms found at subsequent circadian times, implying that multisite phosphorylation events on FRQ modulate the FFC-WCC assembly. Phosphorylation of FRQ may negatively impact WCC's accessibility to certain important D/E residues of FRQ. Alternatively, mutations to these D/E residues may impinge on nearby phosphoevents, although we did not notice obvious alterations of the overall phosphorylation profiles of FRQ in these mutants (Figs. 5 and 6B). Strong defects in FRQ phosphorylation have been noticed in several frq mutants from this study, including frq D773A, D774A (Fig. 6B), frq E760A, E763A, D773A, D774A (the rightmost panel of Fig. 5), frq Δ775-800 (lower left of Fig. 2A), and frq 311-628 (Fig. 7B), which suggest that CK1 does not function normally in phosphorylating FRQ in these frq mutants given that CK1 is a stable component of FFC and also the major kinase in mediating FRQ phosphorylation (See the paragraph of "FRQ functions mainly as…"). In summary, we confirmed that multiple regions of FRQ are required for FFC to bind WCC and identified important negatively charged residues of FRQ required for WCC interaction.

Protein lysate and Western blot
Protein lysates for Western blots (WBs) were prepared as previously described (56,57). Liquid medium for culturing Neurospora is composed of 1× Vogel's, 0.5% arginine, 50 ng/ml biotin, and 2% glucose. Vacuum-dehydrated Neurospora tissue was frozen thoroughly in liquid nitrogen and ground to a fine powder using a mortar and pestle, the protein-extraction buffer (50 mM Hepes [pH 7.4], 137 mM NaCl, 10% glycerol, 0.4% NP-40) with cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Roche, Catalog # 04693159001, at a dilution of one tablet to 10 ml buffer) was added to the powder, and the mixture of the Neurospora tissue powder and buffer was treated with cycles of vortexing for 10 s and resting on ice for another 10 s for a total of 2 min. For WB, equal amounts (15 μg) of centrifugation (12,000 rpm at 4 C for 10 min)cleared whole-cell lysate were loaded per lane in a commercial 3 to 8% 1.5-mm × 15-well Tris-Acetate SDS gel (Thermo Fisher Scientific, Catalog # EA03785BOX) with 1× NuPAGE Tris-Acetate SDS Running Buffer (Thermo Fisher Scientific, Catalog # LA0041). Rabbit V5 antibody (Abcam, Catalog # ab9116), mouse FLAG antibody (Sigma-Aldrich, Catalog # F3165), or rabbit HA (Abcam, Catalog # ab9110) was diluted at 1:5000 as the primary antibodies (48). Custom rabbit FRQ, FRH, WC-1, and WC-2 antibodies have been described previously for applications in WB (6,32,58).
Immunoprecipitation IP with Neurospora lysate was performed as previously described (21). Briefly, 2 mg of total protein (cleaned by 12,000 rpm centrifugation at 4 C for 10 min) were incubated with 20 μl of V5 agarose (Sigma-Aldrich, Catalog #7345) by rotating at 4 C for 2 h. The agarose beads were then washed twice with the same protein extraction buffer (50 mM Hepes [pH 7.4], 137 mM NaCl, 10% glycerol, 0.4% NP-40) and eluted by adding 100 μl of 5 × SDS sample buffer and then being heated at 99 C for 5 min. Ten out of the 100 μl IP were loaded per lane in WB.

Luciferase assays
Luciferase assays were performed as previously described (59,60). 96-well plates with each well containing 0.8 ml of the luciferase-assay medium were inoculated with conidial suspension, and the inoculated strains were grown at 25 C plus constant light for 16 to 24 h and then transferred to the dark at the same temperature for recording the light production. Bioluminescence signals were recorded with a charge-coupled device camera every hour, data were obtained with ImageJ and a custom macro, and period lengths of the strains were manually calculated. Raw data from three replicates were shown in the figures, and time (in hours) is on the x-axis, while arbitrary units of the signal intensity are on the y-axis. Medium for luciferase assays contains 1× Vogel's salts, 0.17% arginine, 1.5% bacto-agar, 50 ng/ml biotin, 0.1% glucose, and 12.5 μM luciferin (GoldBio, Catalog # LUCK-2G). WT used in luciferase assays was 661-4a (ras-1 bd , A) containing the frq C-box fused to the codonoptimized firefly luciferase gene (transcriptional fusion) at the his-3 locus.

Data availability
The Neurospora strains made in this study are available upon request. All data used to draw conclusions of the article have been presented within the figures.